Benzamidoxime prodrugs as antipneumocystic agents

ABSTRACT

A method of treating  Pneumocystis carinii  pneumonia in a subject in need of such treatment is disclosed. The method comprises orally administering to the subject a bis-benzamidoxime, or a pharmaceutically acceptable salt thereof, that is reduced in the subject to produce a benzamidine having anti- P. carinii  activity. The method of the present invention may alternatively comprise intravenously administering to the subject an active compound as disclosed herein. Pharmaceutical formulations and active compounds useful in the practice of the present invention are also disclosed.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/127,317, filed Jul. 31, 1998, now U.S. Pat. No. 6,025,398, which is adivisional of U.S. application Ser. No. 08/751,171, filed Nov. 15, 1996,now U.S. Pat. No. 5,843,980, which is a continuation-in-part of U.S.application Ser. No. 08/558,716, filed Nov. 16, 1995 now U.S. Pat. No.5,723,495.

The present invention was made with Government support under GrantNumber 5-UO1-AI33363-03 from the National Institutes of Health. TheGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to methods useful in combatingPneumocystis carinii pneumonia and prodrug compounds useful therefor.

BACKGROUND OF THE INVENTION

Pentamidine is used for the treatment of Pneumocystis carinii pneumonia,or “PCP”. The importance of pentamidine has dramatically escalatedrecently due to the marked increase of patients suffering from PCP. Theincrease in the afflicted patient population is an unfortunateconsequence of the increasing presence of the Acquired ImmunodeficiencySyndrome (“AIDS”). It is now estimated that approximately 70 percent ofAIDS patients contract PCP. Because of the high incidence of PCP in AIDSpatients, pentamidine has found utility not only in the treatment ofPCP, but also as prophylaxis, in preventing or delaying the initialonset or recurrence of PCP, especially in AIDS patients. Currently,pentamidlne is most commonly administered as a therapeutic agent byintravenous infusion and as a prophylactic agent by aerosol dosage.

However, an unfortunate side effect of pentamidine is its toxicity. Somefatalities have been attributed to severe hypotension, dysglycemia, andcardiac arrhythmias in patients treated with pentamidine. Contrawise,insufficient dosage may result in dissemination of disease beyond thelung, an occurrence which is associated with a poor prognosis.Therapeutic drug monitoring is not used because of the cost andcomplexity of the currently available assay techniques which require theextraction of plasma and High Performance Liquid Chromatography (HPLC)analysis. As a result, the toxicity of pentamidine is a significantconcern, which is driving the market toward the development ofpentamidine substitutes capable of avoiding or minimizing theundesirable side effects associated with the use of pentamidine. See,e.g., J. Spychala et al., Eur. J. Med. Chem. 29, 363-367 (1994); I. O.Donkor et al., J. Med. Chem. 37, 4554-4557 (1994); R. R. Tidwell et al.,J. Protozool. 6, 148S-150S (1991).

Accordingly, it is an object of the present invention to provide newcompounds useful in the treatment of P. carinii pneumonia.

SUMMARY OF THE INVENTION

A method of treating Pneumocystis carinii pneumonia in a subject in needof such treatment is disclosed. The method comprises orallyadministering to the subject a bis-benzamidoxime, derivative thereof, ora pharmaceutically acceptable salt thereof (hereinafter referred to asthe “active compound”), that is reduced in the subject to produce abenzamidine having anti-P. carinii activity. The method of the presentinvention may alternatively comprise intravenously administering to thesubject an active compound as disclosed herein.

A second aspect of the present invention is a pharmaceutical formulationcomprising, in combination with a pharmaceutically acceptable carrier, abis-benzamidoxime, or a pharmaceutically acceptable salt thereof, thatis reduced in a mammalian subject after administration thereto toproduce a benzamidine having anti-Pneumocystis carinii activity, subjectto the proviso that said bis-benzamidoxime is not1,5-bis(4′-(N-hydroxyamidino)phenoxy)pentane.

A third aspect of the present invention are active compounds useful incarrying out a therapeutic method of the present invention.

A fourth aspect of the present invention is the use of an activecompound as disclosed herein for the manufacture of a medicament usefulin carrying out a therapeutic method of treatment as given above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a spectral illustration of the metabolism ofbis-benzamidoximes of the present invention by rat liver homogenate9000×g supernatant fraction. In the metabolism studies illustrated inFIGS. 1A, 1B, 1C, and 1D, homgenates containing 167 μM test compound assubstrate plus a cofactor solution were incubated at 37° C. for 10minutes, then assayed by HPLC as described below in Example 7. In allFigures, “IS” means internal standard. FIG. 1A illustrates themetabolism of compound 1 into its amidine analog pentamidine (indicatedin the Figure by the numeral 2) and its monoamidine-monoamidoximederivative (indicated in the Figure by the numeral 9). FIG. 1Billustrates the metabolism of compound 3 into its amidine analog(indicated in the Figure by the numeral 4). FIG. 1C illustrates themetabolism of compound 5 into its amidine analog (indicated in theFigure by the numeral 6). FIG. 1D illustrates the metabolism of compound7 into its amidine analog (indicated in the Figure by the numeral 8).

FIG. 2 is a graphical illustration of the time course of reduction ofbis-benzamidoxime compound 1 to its monoamidine-monoamidoxime productexpressed by the measure of nmol of product/mg of protein (y-axis value)as a function of time in minutes (x-axis value). The open circlesrepresent data points for the reduction of compound 1 in the rat liverpost-mitochondrial supernatant fraction described below in Example 7.The closed circles represent data points for the reduction of compound 1in the rat liver microsomal fractions, also described in Example 7.

FIG. 3 is a spectral illustration of the metabolism of bis-benzamidoximecompound 1 into its amidine analog pentamidine (indicated in the Figureby the numeral 2) and its monoamidine-monoamidoxime derivative(indicated in the Figure by the numeral 9) by the 9000×g supernatants ofhomogenates of rat liver (FIG. 3A); rat kidney (FIG. 3B); rat lung (FIG.3C) and rat heart (FIG. 3D).

FIG. 4 is a spectral illustration of the metabolism ofbis-benzamidoximes of the present invention in intact BRL 3A hepatocytesin vitro. In the metabolism studies illustrated in FIGS. 4A, 4B, 4C, and4D, cells cultured in Ham's F-12 medium containing 5% fetal bovine serumand 10 μM diamidoxime substrate were incubated for 24 hours at 37° C.under 5% CO₂. The extracellular medium was extracted and then assayed byHPLC as described below in Example 7. In all Figures, “IS” meansinternal standard. FIG. 4A illustrates the metabolism of compound 1 intoits amidine analog pentamidine (indicated in the Figure by the numeral2) and its monoamidine-monoamidoxime derivative (indicated in the Figureby the numeral 9). FIG. 4B illustrates the metabolism of compound 3 intoits amidine analog (indicated in the Figure by the numeral 4). FIG. 4Cillustrates the metabolism of compound 5 into its amidine analog(indicated in the Figure by the numeral 6). FIG. 4D illustrates themetabolism of compound 7 into its amidine analog (indicated in theFigure by the numeral 8).

DETAILED DESCRIPTION OF THE INVENTION

Active compounds of the present invention are, in general, thebis-benzamidoxime derivatives of benzamidines that haveanti-Pneumocystis carinii activity. The benzamidines having anti-P.carinii activity may be mono-benzamidines, wherein one amidoxime groupof the bis-benzamidoxime derivative is reduced; alternatively, they maybe bis-benzamidines wherein both amidoxime groups of thebis-benzamidoxime derivative are reduced. Thus, bis-benzamidoximederivatives of benzamidines having anti-P. carinii activity are anaspect of the present invention. Examples of such benzamidines aredisclosed in, e.g., U.S. Pat. No. 2,277,861 to Ewins et al.; U.S. Pat.No. 2,410,796 to Newberry et al.; U.S. Pat. No. 4,933,347 to Tidwell etal.; and PCT Application No. US93/09477 (applicant specifically intendsthe disclosure of these and all other patent references cited herein tobe incorporated herein by reference).

As used herein, the term “cycloalkyl” as used herein refers to C3 to C6cyclic alkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. Cyclohexyl and cyclopentyl are currently preferred. The term“aryl” as used herein refers to C3 to C10 cyclic aromatic groups such asphenyl, naphthyl, and the like, and includes substituted aryl groupssuch as tolyl. The term “hydroxyalkyl” as used herein refers to C1 to C4linear or branched hydroxy-substituted alkyl, i.e., —CH₂OH, —(CH₂)₂OH,etc. The term “aminoalkyl” as used herein refers to C1 to C4 linear orbranched amino-substituted alkyl, wherein the term “amino” refers to thegroup NR′R″, wherein R′ and R″ are independently selected from H orlower alkyl as defined above, i.e., —NH₂, —NHCH₃, —N(CH₃)₂, etc. Theterm “oxyalkyl” as used herein refers to C1 to C4 oxygen-substitutedalkyl, i.e., —OCH₃, and the term “oxyaryl” as used herein refers to C3to C10 oxygen-substituted cyclic aromatic groups.

One preferred group of compounds useful in the practice of the presentinvention are bis-benzamidoximes of the formula I:

wherein:

R₁ and R₂ are each independently selected from the group consisting ofH, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl,aminoalkyl or alkylaminoalkyl;

R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen;

n is from 2 to 6;

X is O or S; and

Y is H or loweralkyl;

or pharmaceutically acceptable salts thereof.

A second preferred group of compounds useful in the practice of thepresent invention are bis-benzamidoximes of the formula II:

wherein:

R₁ and R₂ are each independently selected from the group consisting ofH, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl,aminoalkyl or alkylaminoalkyl;

R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl,aryl, aminoalkyl, alkylaminoalkyl or halogen;

R₄ is —OY, or R₁ and R₄ together represent

 wherein R₅ is

Y is H or loweralkyl;

n is an integer from 0 to 2; and

A is a heterocyclic aromatic group selected from the group consistingof:

 wherein R₆ and R₇ are each independently selected from the groupconsisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, oroxyarylalkyl;

or pharmaceutically acceptable salts thereof.

As noted above, the methods of the present invention are useful fortreating P. carinii pneumonia. The methods of the present invention areuseful for treating these conditions in that they inhibit the onset,growth, or spread of the condition, cause regression of the condition,cure the condition, or otherwise improve the general well-being of asubject inflicted with, or at risk of contracting the condition.

Subjects to be treated by the methods of the present invention aretypically human subjects, although the methods of the present inventionmay be useful with any suitable subject known to those skilled in theart.

As noted above, the present invention provides pharmaceuticalformulations comprising the aforementioned active compounds, orpharmaceutically acceptable salts thereof, in pharmaceuticallyacceptable carriers for oral, intravenous, or aerosol administration asdiscussed in greater detail below.

The therapeutically effective dosage of any specific compound, the useof which is in the scope of present invention, will vary somewhat fromcompound to compound, patient to patient, and will depend upon thecondition of the patient and the route of delivery. As a generalproposition, a dosage from about 0.1 to about 100 mg/kg will havetherapeutic efficacy, with all weights being calculated based upon theweight of the active base, including the cases where a salt is employed.A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oraladministration. The duration of the treatment is usually once per dayfor a period of two to three weeks or until the P. carinii pneumonia isessentially controlled. Lower doses given less frequently can be used toprevent or reduce the incidence of recurrence of the infection.

In accordance with the present method, an active compound as describedherein, or a pharmaceutically acceptable salt thereof, may beadministered orally as a solid or as a liquid, or may be administeredintravenously. Alternatively, the active compound or salt may also beadministered by inhalation. When administered through inhalation theactive compound or salt should be in the form of a plurality of solidparticles or droplets having a particle size from about 0.5 to about 5microns, preferably from about 1 to about 2 microns.

Besides providing a method for treating P. carinii pneumonia, the activecompounds of the present invention also provide a method for prophylaxisagainst P. carinii pneumonia in an immunocompromised patient, such asone suffering from AIDS, who has had at least one episode of P. cariniipneumonia, but who at the time of treatment is not exhibiting signs ofpneumonia. As P. carinii pneumonia is an especially potentiallydevastating disease for immunocompromised patients it is preferable toavoid the onset of P. carinii pneumonia, as compared to treating thedisease after it has become symptomatic. Accordingly, the presentinvention provides a method for the prophylaxis against P. cariniipneumonia comprising administering to the patient a prophylacticallyeffective amount of the active compound or a pharmaceutically acceptablesalt thereof. The forms for administration of the compound or salt inaccordance with this method may be the same as utilized for the purposeof actually treating a patient suffering from P. carinii pneumonia.

An additional useful aspect of the present invention is a method forprophylaxis against even an initial episode of P. carinii pneumonia inan immunocompromised patient who has never experienced an episode of P.carinii pneumonia. In this respect, a patient who has been diagnosed asbeing immunocompromised, such as one suffering from AIDS or ARC (AIDSrelated complex), even before the onset of an initial episode of P.carinii pneumonia, may avoid or delay suffering from the infection byhaving administered a prophylactically effective amount of an activecompound of the present invention or a pharmaceutically acceptable saltthereof. The compound or salt may be administered in the same fashion asin the treatment of patients suffering from P. carinii pneumonia.

In the manufacture of a medicament according to the invention (a“formulation”), active agents or the pharmaceutically acceptable saltsthereof (the “active compound”) are typically admixed with, inter alia,an acceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the subject. The carrier may be solid orliquid, or both, and is preferably formulated with the compound as aunit-dose formulation, for example, a tablet, which may contain from0.05% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention (e.g.the formulation may contain one or more additional anti-P. cariniiagents as noted above), which formulations may be prepared by any of thewell-known techniques if pharmacy consisting essentially of admixing thecomponents, including one or more accessory therapeutic ingredients.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder. Formulations for oral administration may optionally includeenteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

In addition to the active compounds or their salts, the pharmaceuticalcompositions may contain other additives, such as pH adjustingadditives. In particular, useful pH adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the compositions may contain microbialpreservatives. Useful microbial preservatives include methylparaben,propylparaben, and benzyl alcohol. The microbial preservative istypically employed when the formulation is placed in a vial designed formultidose use. Of course, as indicated, the pharmaceutical compositionsof the present invention may be lyophilized using techniques well knownin the art.

Other pharmaceutical compositions may be prepared from thewater-insoluble active compounds, or salts thereof, such as aqueous baseemulsions. In such an instance, the composition will contain asufficient amount of pharmaceutically acceptable emulsifying agent toemulsify the desired amount of the active compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

Further, the present invention provides liposomal formulations of theactive compounds and salts thereof. The technology for forming liposomalsuspensions is well known in the art. When the active compound or saltthereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the active compounds orsalts thereof, may be lyophilized to produce a lyophilizate which may bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired active compound or a saltthereof or a plurality of solid particles of the compound or salt. Thedesired formulation may be placed in a small chamber and nebulized.Nebulization may be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles should have a particle size in the range of about 0.5 to about5 microns. The solid particles can be obtained by processing the solidactive compound, or a salt thereof, in any appropriate manner known inthe art, such as by micronization. Most preferably, the size of thesolid particles or droplets will be from about 1 to about 2 microns. Inthis respect, commercial nebulizers are available to achieve thispurpose.

Preferably, when the pharmaceutical formulation suitable foradministration as an aerosol is in the form of a liquid, the formulationwill comprise a water-soluble active compound of the present inventionor a salt thereof, in a carrier which comprises water. A surfactant maybe present which lowers the surface tension of the formulationsufficiently to result in the formation of droplets within the desiredsize range when subjected to nebulization.

Formulations of the present invention suitable for intravenousadministration comprise sterile aqueous and non-aqueous injectionpreparations of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay include anti-oxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit/dose or multi-dose containers, for example sealedampules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.

As indicated, the present invention provides both water-soluble andwater-insoluble compounds and salts. As used in the presentspecification, the term “water-soluble” is meant to define anycomposition which is soluble in water in an amount of about 50 mg/mL, orgreater. Also, as used in the present specification, the term“water-insoluble” is meant to define any composition which hassolubility in water of less than about 20 mg/mL. For certainapplications, water soluble compounds or salts may be desirable whereasfor other applications water-insoluble compounds or salts likewise maybe desirable.

Examples of active compounds of the present invention include, but arenot limited to:

1,3-bis(4′-(N-hydroxyamidino)phenoxy)propane

1,3-bis(2′-methoxy-4′-(N-hydroxyamidino)phenoxy)propane

1,4-bis(4′-(N-hydroxyamidino)phenoxy)butane

1,3-bis(4′-(4-hydroxyamidino)phenoxy)propane di-hemimaleinate

1,3-bis(2′-methoxy-4′-(N-hydroxyamidino)phenoxy)propane di-hemimaleinate

1-(4′-(N-hydroxyamidino)phenoxy)butane bis-maleinate

2,5-bis-[4-amidinophenyl]furan bis-amidoxime

2,5-bis-[4-amidinophenyl]furan bis-O-methylamidoxime

2,5-bis-[4-amidinophenyl]furan bis-O-ethylamidoxime

Compounds employed in carrying out the present invention may be preparedin accordance with techniques known to those skilled in the art,particularly in light of the disclosure and examples set forth below.

As indicated, the compounds used in the present invention may be presentas pharmaceutically acceptable salts. Such salts include the maleinate,gluconate, lactate, acetate, tartarate, citrate, phosphate, borate,nitrate, sulfate, and hydrochloride salts.

The salts of the present invention may be prepared, in general, byreacting two equivalents of the pyrimidine base compound with thedesired acid, in solution. After the reaction is complete, the salts arecrystallized from solution by the addition of an appropriate amount ofsolvent in which the salt is insoluble.

As noted above, the active compounds of the present invention accordingto Formula I may be prepared according to methods known in the art. Forexample, the active compounds above may be prepared by firstsynthesizing known bis-nitriles using Allen's procedure for alkylationof phenols. See J. N. Ashley et al., J. Chem. Soc. 103-116 (1942); C. F.H. Allen et al. Org. Synth. Coll. III, 141-41 (1955). The activecompounds can then be obtained by using variations of the knowntechnique of Clement and Raether and by using appropriate reagents. SeeB. Clement and W. Raether, i Arzneim. Forsch. 35, 1009-1014 (1985).

Active compounds of the present invention according to Formula II mayalso be produced by methods known in the art. For example, a two-stepprocess using Pinner methodology may be used to convert a nitrile intoan imidate ester, followed by reaction of the imidate ester withhydroxylamine. A. Pinner, Ber. 17, 184 (1884). Alternatively, amidoximesaccording to Formula II may be prepared by the direct conversion of anitrile into the amidoxime by the reaction with hydroxylamine in thepresence of base. I. Lamb et al., J. Chem. Soc. 1253 (1939).

Subjects with other microbial infections, in addition to P. cariniipneumonia, may also be treated by the methods of the present inventionin the same manner as described above. These infections may be caused bya variety of microbes, including fungi, algae, protozoa, bacteria, andviruses. Exemplary microbial infections that may be treated by themethod of the present invention include, but are not limited to,infections caused by Giardia lamblia, Cryptosporidium parvum,Cryptococcus neoformans, Candida albicans, Candida tropicalis,Salmonella typhimurium, Plasmodium falciparum, and Leishmania mexicanaamazonensis.

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereon. In these examples, mMmeans millimolar, mL means milliliters, mm means millimeters, cm meanscentimeters, °C. means degrees Celsius, g means grams, kg meanskilograms, m.p. means ng point, MHz means megahertz, M means molar, hhours, NMR means nuclear magnetic resonance, FAB fast atom bombardment,DMF means dimethylformamide, EtOH means ethyl alcohol, DMSO meansdimethylsulfoxide, HPLC means high-pressure liquid chromatography, TLCmeans thin-layer chromatography, dec means decomposition point.

In the following Examples, the following compound designations are usedthroughout.

Compound # Name a 1,5-bis(4′-(N-hydroxyamidino)phenoxy)pentane b1,3-bis(4′-(N-hydroxyamidino)phenoxy)propane c1,3-bis(2′-methoxy-4′-(N-hydroxyamidino)phenoxy)propane d1,4-bis(4′-(N-hydroxyamidino)phenoxy)butane 11,5-bis(4′-(N-hydroxyamidino)phenoxy)pentane di-hemimaleinate 21,5-bis(4′-amidinophenoxy)pentane; pentamidine 31,4-bis(4′-(N-hydroxyamidino)phenoxy)butane di-maleinate 41,4-bis(4′-amidinophenoxy)butane 51,3-bis(4′-(4-hydroxyamidino)phenoxy)propane di-hemimaleinate 61,3-bis(4′-amidinophenoxy)propane 71,3-bis(2′-methoxy-4′-(N-hydroxyamidino)phenoxy)propane di-hemimaleinate8 1,3-bis(2′-methoxy-4′-amidinophenoxy)propane 92,5-bis-[4-amidinophenyl]furan 10  2,5-bis-[4-amidinophenyl]furanbis-amidoxime 11  2,5-bis-[4-amidinophenyl]furan bis-O-methylamidoxime12  2,5-bis-[4-amidinophenyl]furan bis-O-ethylamidoxime

EXAMPLE 1 Synthesis of Formula I Compounds: Preparation ofbis-benzonitriles

42 mmol of 1,5-dibromopentane (for preparing pentamidine derivatives) or1,3-dibromopentane (for preparing propamidine derivatives) is added to asuspension of 84 mmol of the appropriate 4-hydroxybenzonitrile and 126mmol of K₂CO₃ in 200 mL DMF. The mixture is warmed to 65-70° C. andallowed to stir overnight. The mixture is diluted in 400 mL of H₂O, theprecipitated product is collected and washed with H₂O. The crudebis-benzonitriles are recrystallized from ethanol.

EXAMPLE 2 Synthesis of Formula I Compounds: Preparationbis-benzamidoximes

32 mL of a 21% sodium ethoxide (in ethanol) solution is added to a hotsolution of 98 mmol of NH₂OH.HCl in 100 mL ethanol. The NaCl is removedby filtration and the filtrate is entered directly into a flaskcontaining 10 mmol of the appropriate bis-benzonitrile from Example 1.The mixture is warmed to reflux and allowed to stir for 5 hrs, cooled toroom temperature and permitted to stand overnight. The precipitatedproduct is collected, washed with ethanol and dried in a vacuumdesiccator. The following spectral and analytic data were collected:

Compound (a): m.p.>164-65° C.; (literature value: 163° C.; see R. R.Tidwell et al., J. Med Chem. 26, 294-98 (1983)); 3.6 g, 60%.

Novel Compound (b): m.p. 162° C., (1.6 g, 47%); ¹H-NMR (300 MHz, DMSO)δ2.18 (m, 2 H), 4.16 (t, J=5.9 Hz, 4 H), 5.72 (s, 4 H), 6.95 (d, 4 H,J=8.6 Hz), 7.59 (d, 4 H, J=8.6 Hz), 9.45 (s, 2 H) ppm; FABMS m/z 345(M+H); Exact mass calculated for C₁₇H₂₁N₁₄O₄: 345.1563; found: 345.1557;Anal. (C₁₇H₂₁N₁₄O₄) C, H, N.

Novel Compound (c): m.p.117° C. (2.9 g, 73%); ¹H-NMR (300 MHz, DMSO)δ2.17 (m, 2 H), 4.14 (t, 4 H, J=5.9 Hz), 5.74 (s, 4 H), 6.98 (d, 2 H,J=8.4 Hz), 9.46 (s, 2 H) ppm; FABMS m/z 405 (M+H); Exact mass calculatedfor C₁₉H₂₅N₄O₆: 405.1774; found: 405.1795; Anal.(C₁₉H₂₅N₄O₆.(H₂O)_(1.3): C, H, N.

Novel Compound (d): m.p.200° C. (dec) (1.0 g, 33%); ¹H-NMR (300 MHz,DMSO) δ1.87 (s, 2 H), 4.05 (s, 2 H), 5.72 (s, 4 H), 6.95 (d, 4 H, J=8.7Hz), 7.60 (d, 2 H, J=8.7 Hz), 9.45 (s, 2 H) ppm; FABMS m/z 359 (M+H);Anal. (C₁₈H₂₂N₄O₄): C, H, N.

Further elemental analysis data is shown in Table 1.

TABLE 1 Elemental Analyses of Novel Amidoximes Compound MolecularFormula Calculated Found b C₁₇H₂₀N₄O₄ C: 59.30; H: 5.81; C: 59.12; H:5.86; N: 16.28 N: 16.00 c C₁₉H₂₄N₄O₆.(H₂O)_(1.3) C: 53.34; H: 6.27; C:53.64; H: 6.01; N: 13.10 N: 12.71 d C₁₈H₂₂N₄O₄ C: 59.13; H: 6.29; C:59.75; H: 6.24; N: 15.32 N: 14.67 3 C₁₈H₂₂N₄O₄.1.60C₄H₄O₄ C: 58.21; H:6.65; C: 57.99; H: 6.55; N: 12.93 N: 13.29 5 C₂₅H₂₈N₄O₁₂.(H₂O)_(1.6) C:49.59; H: 5.20; C: 49.39; H: 4.99; N: 9.26 N: 9.59 7 C₂₇H₃₂N₄O₁₄.(H₂O)C: 49.53; H: 5.26; C: 53.88; H: 5.31; N: 10.30 N: 10.03

EXAMPLE 3 Synthesis of Formula I Compounds: Preparation of MaleinateSalts of bis-benzamidoxime Compounds

2.7 mmol of the appropriate bis-amidoxime of Example 2 was taken up in20 mL hot THF. Insoluble impurities (monoadducts) are removed byaddition of 20 mL of a THF solution of either 6.8 mmol or 3.6 mmol ofmaleic acid. The resulting precipitate is collected and washed withether/ethanol and then ether to give the bis-hemimaleinates as colorlesspowders in most cases. The following analytical and spectral data areobserved:

Compound (1): m.p. 118-120° C. (literature 115-120° C., see R. R.Tidwell et al., J. Med Chem. 26, 294-98 (1983)), (1.2 g, 75%).

Novel Compound (3): m.p. 154° C. (1.1 g, 67%) Anal. [C₁₈H₂₂N₄O₄ (C,H,N).

Novel Compound (5): m.p. 135° C. (dec) (800 mg, 91%); Anal.(C₂₅H₂₈N₄O₁₂.(H₂O)_(1.6)) C,H,N.

Novel Compound (7): m.p. 134° C. (dec) (700 mg, 86%) Anal.[C₂₇H₃₂N₄O₁₄.(H₂O)] C, H, N.

Further elemental analysis data is shown in Table 1.

EXAMPLE 4 Activity of Novel Compounds of Formula I Against Pneumocystiscarinii

The activity of the compounds of Formula I against P. carinii wascarried out according to an established method. See Tidwell, R. R., etal., J. Protozool. 36, 74S-76S (1989); Jones, S. K., et al., Antimicrob.Agents Chemother. 34, 1026-1030 (1990); Tidwell, R. R., et al.,Antimicrob. Agents Chemother. 37, 1713-1716 (1993). Briefly, maleSprague-Dawley rats (barrier raised, not certified virus-free) weighing150-200 g each, were obtained (Hilltop Laboratories, Scottsdale, Pa.).Immediately upon arrival, the animals were caged individually and begunon a low protein (8%) diet and on drinking water containing tetracycline(0.5 mg/mL) and dexamethasone (1.0 μ/mL). This regimen was continued foreight weeks. At the beginning of the seventh week, animals were dividedinto groups of 8 or more, and the test compounds were administered for14 days. Saline and pentamidine-treated groups were included ascontrols.

Animals were sacrificed at the end of the eighth week by chloroforminhalation. The left lung was weighed, ground through a No. 60 wire meshscreen, and suspended 1:10 (wt/vol) in 10 mM β-mercaptoethanol-Hanks'balanced salts solution (HBSS) without cations. Slides were prepared byspotting 5 μL of lung homogenate diluted 1:10 in HBSS withβ-mercaptoethanol and allowed to air dry. The slides were stained withcresyl violet, and the cysts were counted by a blinded protocol. Thenumber of cysts per gram of original lung tissue was calculated, and thegroups were reported as the percentages of saline-treated controls.

The anti-P. carinii value for each compound is expressed as the percentof cysts in the treatment group as compared to the saline control group.Values are also compared to a positive control group consisting ofanimals treated intravenously with pentamidine.

All four aromatic amidoximes tested were active against P. carinii whenadministered orally by gavage once daily for 14 days (see Table 2). Meancyst counts for each test group were significantly reduced compared tothe saline control group. Compounds 3, 5 and 7 were most active. Thearomatic diamidines corresponding with the amidoximes 3, 5, and 7(compounds 4, 6 and 8, respectively), in contrast, had reduced or noanti-P. carinii activity when given orally (see Table 3). The onlydiamidine with significant oral activity was compound 8. Itscorresponding diamidoxime (compound 7) was more active (see Table 2).

All aromatic amidoximes tested had excellent anti-P. carinii activitywhen given intravenously. Mean cyst counts for Compounds 1, 3, 5, and 7were significantly reduced compared to saline controls (see Table 2),and were also lower than cyst counts for the intravenous pentamidinegroup. The diamidine compounds 4, 6, and 8 were previously shown to haveintravenous activity (see Table 3). Direct comparisons of diamidine andcorresponding diamidoxime (Compounds 3, 5, 7) intravenous activitiescannot be made, however, as the intravenous activities of diamidineswere previously evaluated by a different score method and at slightlydifferent doses. However, the data indicates that the amidoximes comparefavorably with regard to intravenous efficacy.

EXAMPLE 5 Novel Compounds of Formula II Antipneumocystic Activity andToxicity

It has been reported that 2,5-bis-[4-amidinophenyl]furan (compound 9) ismore active and less toxic than pentamidine against Pneumocystis cariniiin the immunosuppressed rat model on intravenous dosage. D. W. Boykin etal., J. Med. Chem. 38, 912 (1995). However, the activity of 9 in the ratmodel was significantly less on oral administration.

The aromatic amidoxime derivatives of compound 9, listed above ascompounds 10, 11, and 12, were prepared according to a two-step processusing Pinner methodology to convert a nitrile to an imidate ester,followed by reactions of the imidate ester with hydroxylamine. A.Pinner, Ber. 17, 184 (1884).

Table 4 provides the results of evaluation of the prodrugs 10, 11, and12, administered as dimaleinate salts, against P. carinii pneumonia inthe immunosuppressed rat model (see Example 4) on both oral andintravenous administration. Data from compound 9 are provided forcomparison purposes. In Table 4 the following legend is applicable ininterpreting the data:

^(a) dosage by tail vein injections

^(b) subjective scale of toxicity. See R. R. Tidwell, et al.,Antimicrob. Agents Chemother. 37, 1713 (1993). In general, the largerthe value the more severe the toxicity, with values greater than 2indicating the death of some animals

^(c) cysts counted in lung tissue employing a blinded protocol, andreported as a percentage of the saline treated controls. See R. L.Lombardy et al., J. Med. Chem. 39, 1452 (1996).

^(d) oral dosage by gavage

^(e) dosage by tail vein injection

^(f) not available, experiment suspended on day 8 due to severe necrosisof the tail at the injection site

^(g) compounds administered as the dimaleinate salt

The results shown in Table 4 demonstrate that compounds 10 and 11 areeffective when given orally by gavage, as compared to the salinecontrol. The data also show that the amidoximes can function as prodrugsin the furan series. The values for the cyst counts suggest that druguptake on oral dosage for 10 and 11 are superior to that of the parentcompound 9. Unexpectedly, the bis-O-methylamidoxime 11 is more effectivethan the bis-amidoxime 10.

On intravenous administration of both 10 and 11, the acute toxicitygenerally noted for diamidines was absent. However, for compound 10,considerable inflammation and necrosis of the tail vein at the injectionsite is observed even at the relatively low dosage of 5.5 μmol/day. Incontrast, the bis-O-methylamidoxime 11 is well-tolerated, asinflammation and necrosis at the injection site are absent even withintravenous administration of 22.0 μmol/kg/day.

EXAMPLE 6 Reduced Acute Toxicity of Amidoximes of Formula I in NormalRats

Toxicity was studied in rats that were not immunosuppressed bydexamethasone treatment. Adult male Sprague-Dawley rats, barrier raised,not certified virus free, and weighing 300 to 450 g at the time, wereobtained from Hilltop Laboratories (Scottsdale, Pa.). The individuallycaged animals were given water and rat chow (Agway, Syracuse, N.Y.) adlibitum. Each animal was injected via the tail vein with one dose oftest compound. Animals were observed closely for a 10 to 12-minuteperiod following injection of the test drug each day for signs of acutetoxicity, including the hypotensive response (paling of eyes and paws,dyspnea, lethargy and decreased body temperature) elicited byintravenous pentamidine at its effective dose. See R. R. Tidwell et al.,J. Protozool. 36, 74S-76S (1989); R. R. Tidwell et al., J.Med. Chem. 33,1252 (1990)). Each animal was closely observed for 15 minpost-injection, and monitored again at 30 min., 60 min. and 24 hr.post-injection. The rats' health and general well-being were observedand recorded on a daily basis for the remainder of the experiment.Excessive weight loss (a greater than 25 g weight loss over the two-weekdosing period was considered excessive in comparison with salinecontrols) was considered a key indicator of declining health due to drugtoxicity. At necroscopy, the liver, spleen, kidneys and pancreas wereremoved from each animal, examined for gross pathology, and saved forhistopathology.

In these normal rats, overt acute adverse reactions following singleintravenous injections were greatly reduced for three bis-benzamidoximes(compounds 1, 5, and 7) as compared to the bis-benzamidines (compounds 2(pentamidine), 6, and 8). No adverse reactions were observed after veryhigh single oral doses of the bis-benzamidoximes. To compare, normalrats injected over a 30-second period with 20 μmol/kg pentamidineappeared hypotensive, with rapid paling of extremities, hypoactivity anddyspnea, which progressed to slight cyanosis of extremities. Increasedlacrimation and minor hind leg ataxia were observed immediately beforeonset of hypoactivity. The animals appeared to recover within 5 minutesafter injection. Animals injected with 40 μmol/kg pentamidine hadimmediate sever hind limb contractions, increased salivation, dyspnea,initial paling of extremities which progressed to marked cyanosis andprofound hypoactivity, with no movement for at least 5 minutes. Theanimals appeared to recover approximately 20 minutes after injection. Incontrast, the diamidoxime analog of pentamidine (compound 1), given inthe same manner, caused no observable adverse reactions from 20 to 60μmol/kg. Minor toxic responses, including barely observable hindlegataxia and slight hypoactivity, were observed at 80 μmol/kg, withcomplete recovery at 5 minutes post-injection. Bolus injections above120 μmol/kg produced severe dyspnea and profound hypoactivity.

Similar results were observed for diamidoxime compounds 5 and 7 comparedwith their respective diamidine analogs. The diamidine compound 6appeared less acutely toxic than pentamidine, with no overt toxicity at20 μmol/kg. Dyspnea progressing to cyanosis, excessive salivation andlacrimation, and hypoactivity, were observed at 40 μmol/kg. Theseresponses became more severe at 80 and 100 μmol/kg, with symptomspersisting for 30 minutes after injection. The diamidoxime compound 5,in contrast, caused no overt toxicity at 20, 40 or 80 μmol/kg, and onlyminor ataxia and/or hypoactivity at the high dose of 160 μmol/kg.Animals had fully recovered at 5 minutes after injection.

The diamidine compound 8 appeared even more acutely toxic than the othertwo diamidines tested, with perceptible hypoactivity occurring inanimals dosed at 10 μmol/kg. Strong muscular contractions and tremors,extensive salivation and lacrimation, dyspnea and marked hypoactivity,which persisted for longer than 15 minutes, were observed in animalsinjected with 20 μmol/kg. In contrast, animals injected with thediamidoxime analog, compound 7, showed no observable adverse effects at20 or 40 μg/kg and only very minor hypoactivity and dyspnea at 80μmol/kg, with recovery at 5 minutes. Compound 7 injected at 160 μmol/kgcaused pronounced hypoactivity, dyspnea, cyanosis, and increasedsalivation and lacrimation, with symptoms persisting for approximately10 minutes after injection.

No overt toxic responses were seen when the rats were given any of thetest compounds orally, including single oral doses as high as 160μmol/kg for each of the diamidoxime compounds 1, 5, and 7.

EXAMPLE 7 In Vitro Metabolism of Compounds of Formula I

In vitro metabolism of diamidoximes 1, 3, 5, and 7 by rat liverhomogenate 9000×g supernatants, post-mitochondrial 105,000×gsupernatants or microsomal fractions were performed as previouslydescribed in B. J. Berger et al., Antimicrob. Agents and Chemotherapy36, 1825-1831 (1992); B. J. Berger et al., J. Pharmacol. ExperimentalTherapeutics 256, 883-89 (1991). Briefly, adult male Sprague-Dawleyrats, barrier raised, (Hilltop Laboratories, Scottsdale, Pa.) animalswere given water and rat chow (Agway, Syracuse, N.Y.) ad libitum. Therats were euthanized by decapitation, the livers removed immediately,rinsed with 50 mM potassium phosphate buffer, pH 7.4, and placed on ice.All subsequent steps were performed at 4° C. The livers were minced,homogenized, and 9000×g supernatants, 105,000×g or 105,000×g microsomalpellets prepared. Each fraction was assayed for protein content asdescribed in M. M. Bradford, Anal. Biochem. 72, 248-254 (1976) andstored at −80° C. Fractions from the rat kidneys, lungs, hearts andbrains were prepared similarly.

Reaction mixtures consisted of 1.5 mL 50 mM potassium phosphate buffer,pH 7.4, 0.5 mL cofactor solution (2 mg/mL NADPH, 1.6 mg/mL MgCl2, 1.04mg/mL glucose-6-phosphate and 2 units/mL glucose-6-phosphatedehydrogenase in 50 mM potassium phosphate buffer pH 7.4), 0.5 mL tissuehomogenate and 0.5 mL of the appropriate diamidoxime substrate at aconcentration of 167 μM. Reactions were started by adding substrate,then mixtures were incubated at 37° C. in a shaking water bath for timesshown in the figures. Reactions were terminated by extracting over C₁₈cartridges and assaying as described below.

Metabolic experiments with intact cultured cells were performed usingthe BRL 3A hepatocyte line, obtained from the UNC-Chapel Hill LinebergerTissue Culture Facility. Cells were routinely cultured in Costar(Cambridge, Mass.) 25 cm² of tissue culture flasks at 37° C. under moist5% CO₂ and 95% air in Ham's F-12 medium (Gibco, Gaithersburg, Md.)containing 5% fetal bovine serum (HyClone Laboratories Inc., Logan,Utah). Confluent cultures were treated with 0.25% trypsin solution(Sigma Chemical Co., ST. Louis Mo.), then approximately 2×10⁵ cells/wellsubcultured into Costar 6-well tissue culture chambers and the cellsallowed to grow to confluency. Ten mL fresh medium was added to eachculture well and incubations were started by adding 100 μL amidoximesolution (prepared in sterile water) to a final concentration of 10 μM.Cell cultures were incubated at 37° C. under moist 5% CO₂ and 95% airfor 24 hours, then aliquots of culture supernatants extracted andassayed for metabolites as described below. Similar metabolicexperiments were performed with cultured J774 A.1 mousemonocyte-macrophage cells cultured in DMEM F-12 medium containing 10%fetal bovine serum and H9c2 rat heart myoblast cells cultured in DMEM Hmedium containing 10% fetal bovine serum.

Samples were extracted using solid phase extraction and assayed usinghigh performance liquid chromatography (HPLC) by methods similar tothose previously described in Berger et al. (1992) and Berger et al.,(1991), supra. Briefly, samples spiked with2,5-bis[4-(N-isopropylamidino)phenyl]furan dihydrochloride as theinternal standard (IS) were extracted over activated C₁₈ Bond Elutcartridges (Varian Associates, Sunnydale, Calif.), washed with water,100% acetonitrile, then eluted with 75% acetonitrile/25% watercontaining 15 mM triethylamine and 35 mM acetic acid. The diamidoximesubstrates elute in the 100% acetonitrile phase, while mono-amidine anddiamidine products elute in the acetonitrile/water mixture containingtriethylamine and acetic acid. Elutes were evaporated to dryness at 40°C. under a gentle stream of nitrogen and resuspended in HPLC gradewater.

Compounds were resolved using a Hewlett-Packard (Avondale, Pa.) model1090 HPLC equipped with a HP 1050 variable wavelength detector set at265 nm, a 4.6×250 mm Zorbax RX diisopropyl C8 column (Mac-Mod, Chadd'sFord, Pa.) maintained at 40° C. and a Vectra 486/66U computer with HPChemstations software. The mobile phase consisted of 15 mM triethylamineand 35 mM acetic acid in HPLC grade water for pump A, and 15 mMtriethylamine and 35 mM acetic acid in 75% acetonitrile in water forpump B. The solvent flow rate was 1.5 mL/min, and the solvent gradientran from 0% B to 25% B at 22 min, to 40% B at 25 min, then 90% B at 35min. All solvents and reagents used for the assays were HPLC grade.Quantities of metabolites formed were calculated, using peak arearations of authentic standard, from standard addition curves generatedby spiking standards into tissue homogenates or tissue culture medium.

It has been shown that rat liver homogenate 9000×g supernatants readilyreduce the diamidoxime analog of pentamidine (compound 1), forming largequantities of the monoamidine-monoamidoxime derivative, and smalleramounts of pentamidine (compound 2). B. Clement et al., Drug Metabol.Dispos. 22, 486-97 (1994); see FIG. 1A. The metabolites have beenpreviously identified by mass spectrometry. U. Bronner et al.,Pharmacol. Toxicol., 77, 114-20 (1995). The bis-benzamidoximes of thepresent invention (compounds 3, 5, and 7), appear to be metabolizedsimilarly by rat liver homogenate 9000×g supernatants, as shown in FIGS.1B, 1C, and 1D, respectively. In each case, two new product peaks wereeluted. The smaller peaks in FIGS. 1B, 1C, and 1D coeluted with thediamidine standard compound 4, 6, and 8, respectively. Althoughsynthetic standards are not available for the monoamidine/monoamidoximederivatives of compounds 3, 5, and 7, the relative retention times ofthe chromatographic peaks are entirely consistent with those predictedfor the monoamidoxime derivatives.

The N-hydroxylation of aromatic amidines to form aromatic amidoximes isknown to be catalyzed by specific cytochromes P450. See B. Clement etal., Drug Metabol. Dispos. 22, 486-97 (1994). However, reduction ofamidoximes back to the amidines has been reported to be catalyzed by anon-cytochrome P450 dependent reductase activity. Id. The data presentedin FIGS. 2 and 3 indicate that reductase is present in thepost-mitochondrial microsomal fraction. However, reductase activityappears to be even higher in the post-mitochondrial supernatant fraction(FIG. 2). The results also show that high reductase activity is alsofound in homogenates from rat kidneys and lung, as well as from heartand brain (FIG. 3). The latter tissues are known to contain littlecytochrome P450 activity.

Finally, intact cells of the established liver cell line BRL 3A (FIG.4), the heart cell line H9c2 (data not shown) and the macrophage cellline J774 A.1 (data not shown) all readily absorbed and metabolized thediamidoxine compounds. These data indicate that metabolism of amidoximeprodrugs is catalyzed by activities other than cytochromes P450, anddemonstrate that tissues and other cells other than liver possess highlevels of activity. It appears that in order for diamidoxime prodrugs tobe effective against extracellular parasites such as P. carinii, thecompounds must apparently enter the host cells, be chemically reducedback to the active amidine analog, then released extracellularly andtaken up by the infectious organism. The process of cell uptake,metabolism, and release, occurs readily in BRL 3A hepatocytes culturedin vitro (FIG. 4). Each of the orally active bis-benzamidoximes 1, 3, 5,and 7 were metabolized predominantly to the corresponding diamidine,which was then released in high concentrations back into the culturesupernatants.

TABLE 2 Activity of aromatic amidoximes against Pneumocystis cariniipneumonia.

ORAL DOSING^(a) IV DOSING^(b) cysts/g lung ± cysts/g lung ± Compound n RS. E. (× 10⁶) % saline # rats S. E. (× 10⁶) % saline # rats Saline — —37.78 ± 9.49 100.00 6 32.36 ± 11.03 100.00 6 Pentamidine — — 50.28 ±15.20 133.09 6  0.28 ± 0.08^(c) 0.87 6 1 5 H  7.28 ± 5.22^(c) 19.27 5 0.05 ± 0.01^(c) 0.15 6 3 4 H  0.54 ± 0.23^(c) 1.43 5  0.04 ± 0.02^(c)0.12 6 5 3 H  0.85 ± 0.36^(c) 2.25 5  0.01 ± 0.003^(c) 0.03 6 7 3 OCH³ 1.40 ± 1.18^(c) 3.71 6  0.01 ± 0.002^(c) 0.03 3 ^(a)Oral dosesadministered daily for 14 days by gavage @33 μmol/kg body weight.^(b)Intravenous doses administered daily for 14 days by tail veininjection at 22 mg/kg body weight. ^(c)Significantly different fromsaline control group (P < 0.05); Student's test.

TABLE 3 Activity of aromatic amidines against Pneumocystis cariniipneumonia.

ORAL DOSING^(a) cysts/g lung ± IV DOSING^(b) Compound n R S. E. (× 10⁶)% saline # rats mean histological score^(c) # rats Saline — — 37.78 ±9.49 100.00 6 3.2 72 Pentamidine 5 H  50.28 ± 15.20 133.09 6 1.1 63 4 4H 17.39 ± 7.03 46.03 6 0.5 8 6 3 H 16.43 ± 6.36 43.49 6 0.9 8 6 3 OCH³ 3.20 ± 0.58 8.47 6 0.6 8 ^(a)Oral doses administered daily for 14 daysby gavage @33 μmol/kg body weight. ^(b)Intravenous doses administereddaily for 14 days by tail vein injection @ 10 mg/kg body weight.Compound 10 given at 5 mg/kg. ^(c)Data reprinted from J. Med. Chem.33:1252 (1990). The histological score was determined subjectively fromcysts detected in stained lung sections. Scores range from a lowinfection score of 0.5 to a high of 4.0.

TABLE 4 Activity of prodrugs of Bis-2,5-[4-Amidophenyl]furan againstPneumocystis carinii pneumonia.

ORAL DOSING IV DOSING Dosage^(a) cysts in lung Dosage^(d) Cysts in LungCompound R (μmol/kg/day) Toxicity^(b) tissue^(c) (μmol/kg/day) Toxicitytissue saline — not done 0 100.0 ± 9.51   2^(e) — not done 22.0 +2 3.94± 1.49  9^(g) H 66.3 0 25.2 ± 9.84 26.6 +1 NA′ 39.8 0 42.3 ± 12.6 13.3 00.79 ± 0.34 2.7 0 7.26 ± 3.56 0.3 0 6.92 ± 3.36 0.03 0 26.3 ± 5.0 10^(g) OH 33.0 0  3.1 ± 1.31 22.0 +1 0.67 ± 0.46 11.0 0 197.6 ± 64.4 11.0 +1 1.37 ± 0.61 5.5 0 127.0 ± 48.4  5.5 +1 42.3 ± 34.4 2.7 0 54.4 ±27.4 0.3 0 203.6 ± 56.4  11^(g) OCH₃ 33.0 0 1.82 ± 0.53 22.0 0 14.3 ±5.14 22.0 0 12.0 ± 11.5 11.0 0 34.7 ± 21.2 5.5 0 264.2 ± 72.9  12^(g)OCH₂CH₃ 33.0 0 121.5 ± 48.4  22.0 +1 104.7 ± 49.9 

The foregoing is illustrative of the present invention and is not to beconstrued to be limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included herein.

That which is claimed is:
 1. A bis-benzamidoxime of Formula I:

wherein: R₁ and R₂ are each independently selected from the group consisting of H, loweralkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; n is from 2 to 6; X is O or S; and Y is H or loweralkyl; or a pharmaceutically acceptable salt thereof, subject to the proviso that said compound of Formula I is not 1,5-bis(4′-(N-hydroxyamidino)phenoxy) pentane.
 2. A bis-benzamidoxime according to claim 1, wherein n is 3 or 4; X is O; Y is H; R₁ and R₂ are each H; and R₃ is H or —OCH₃.
 3. A pharmaceutical formulation comprising, in combination with a pharmaceutically acceptable carrier, a bis-benzamidoxime, or a pharmaceutically acceptable salt thereof, that is reduced in a mammalian subject after oral administration thereto to produce a benzamidine having anti-Pneumocystis carinii activity, subject to the proviso that said bis-benzamidoxime is not 1,5-bis(4′-(N-hydroxyamidino)phenoxy)pentane, wherein said bis-benzamidoxime is of Formula I:

wherein: R₁ and R₂ are each independently selected from the group consisting of H, lower alkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; n is from 2 to 6; X is O or S; and Y is H or lower alkyl.
 4. A pharmaceutical formulation according to claim 3 comprising a maleinate salt of said bis-benzamidoxime of Formula I.
 5. A pharmaceutical formulation according to claim 3, wherein n is 3 or 4; X is O; Y is H; R₁ and R₂ are each H; and R₃ is H or —OCH₃.
 6. A method of treating Pneumocystis carinii pneumonia in a subject in need of such treatment, comprising orally administering to said subject a compound, wherein said compound is a bis-benzamidoxime that is reduced in said subject to produce a benzamidine having anti-P. carinii activity, and wherein said compound is a compound of Formula I:

wherein: R₁ and R₂ are each independently selected from the group consisting of H, lower alkyl, oxyalkyl, alkoxyalkyl, cycloalkyl, aryl, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; R₃ is H, loweralkyl, oxyalkyl, alkoxyalkyl, hydroxyalkyl, cycloalkyl, aryl, aminoalkyl, alkylaminoalkyl or halogen; n is from 2 to 6; X is O or S; and Y is H or lower alkyl; or a pharmaceutically acceptable salt thereof, in an amount effective to treat Pneumocystis carinii pneumonia.
 7. A method according to claim 6, wherein said compound is a maleinate salt of a compound of Formula I.
 8. A method according to claim 6, wherein n is 3 or 4; X is O; Y is H; R₁ and R₂ are each H; and R₃ is H or —OCH₃.
 9. The method according to claim 6, wherein said subject is afflicted with Pneumocystis carinii pneumonia.
 10. The method according to claim 6, wherein said subject is at risk of developing Pneumocystis carinii pneumonia and said compound is administered in a prophylactically effective amount. 